Approximately one-third of all the proteins in eukaryotes enter the ER for post-translational processing and folding. The quality of protein folding is monitored by the ER membrane-resident kinase-ribonuclease Ire1, which is activated by misfolded proteins. Ire1 initiates a non-spliceosomal cytoplasmic splicing reaction of transcription factor encoding mRNA initiating a genome-scale transcriptional program termed unfolded protein response (UPR). Translation of the spliced mRNA yields a UPR-specific transcription factor, termed Hac1 (Cox, J. S. et al., Cell 87:391-404 (1996)) in yeasts and Xbp1 (Yoshida, H. et al., Cell 107:881-91 (2001)) in metazoans, that activates genes involved in protein biogenesis and restores protein folding in the ER. The UPR activates in cancers (Koong, A. C. et al., Cancer Biol Ther 5 (2006); Ma, Y. et al., Nat Rev Cancer 4:966-77 (2004)), in Alzheimer's disease (Kudo, T. et al. Ann NY Acad Sci 977:349-55 (2002)), and in a variety of other cellular anomalies (Zheng, Y. et al. J Microbiol 43:529-36 (2005); Naidoo, N. et al., J Neurochem 92:1150-7 (2005); Doody, G. M. et al., Eur J Immunol 36:1572-82 (2006)), suggesting numerous possible links between abnormal Ire1 activation and cellular dysfunctions.
During the UPR, the ER-lumenal domain (LD) acts as a sensor of unfolded proteins and promotes lateral self-association of Ire1 in the plane of the ER membrane (FIG. 1A). Notably, the purified LD crystallizes as an oligomer that has two distinct crystallographic interfaces. Ire1 surface residues on both interfaces contribute to Ire1 activation in vivo (Credle, J. J. et al., Proc Natl Acad Sci USA 102:18773-84 (2005)). This finding explains an early observation of oligomerization of Ire1 during the UPR (Shamu, C. E. et al., Embo J 15:3028-39 (1996)) and provides a first structural rationalization of Ire1 organization in UPR-induced foci that can be observed by life-cell imaging (Kimata, Y. et al. J Cell Biol 179:75-86 (2007)) (Aragon et al., 2008). It has been proposed that oligomerization of the LD would increase the local concentration of the kinase-RNase domains of Ire1 on the cytosolic side of the ER membrane and activate the enzymatic domains by dimerization (Credle, J. J. et al., Proc Natl Acad Sci USA 102:18773-84 (2005)). This mechanism of activation parallels that for many well-understood cell surface signaling receptors. Ligand-induced dimerization of epithelial growth factor receptors (EGFR) (Zhang, X. et al., Cell 125:1137-49 (2006)), for example, activates the kinase domains by inducing conformational changes that include opening of the N- and the C-lobes of the kinase and rearrangement of the activation loop and the highly conserved αC helix (Zhang, X. et al., Cell 125:1137-49 (2006)). In addition to self-association, activation of Ire1 involves autophosphorylation and binding of ADP as a co-factor. Both of these events are thought to facilitate a conformational change that activates the RNase (Papa, F. R. et al., Science 302:1533-7 (2003); Gonzalez, T. N. et al., Methods Mol Biol 160:25-36 (2001)).
A crystal structure of the Ire1 kinase-RNase domain has been reported (Lee, K. P. et al., Cell 132:89-100 (2008)). The structure revealed a two-fold symmetric dimer with a back-to-back arrangement of the kinase domains, compactly attached to an RNase dimer with two independent active sites. The structure is well ordered except for the activation loop, the loop following the αD helix of the kinase domain, and a functionally important and apparently highly dynamic loop of the RNase domain. The back-to-back arrangement of the kinases in the dimer is unexpected because it positions the phosphorylation sites in the activation loops 43-48 Å away from the active site of the partnering molecule in the dimer. This arrangement does not appear productive for the trans-autophosphorylation of Ire1 observed in vivo (Shamu, C. E. et al., Embo J 15:3028-39 (1996)) and in vitro (Lee, K. P. et al., Cell 132:89-100 (2008)). The dimerization of the RNase domains has been proposed to allow recognition of the conserved tandem stem-loops comprising the splice sites in HAC1/XBP1 mRNA (Lee, K. P. et al., Cell 132:89-100 (2008)) (FIG. 1B).
The association of ER stress with diverse human diseases, such as cancer, diabetes, proteinopathies, and viral infections, provides reasoning to alter pathogenesis by manipulating the UPR. For example, in cystic fibrosis, it would be beneficial to increase protein folding capacity to produce more chloride channels displayed on the cell surface. On the other hand, in diabetes, pancreatic islet cells die of UPR induced apoptosis, and the Ire1 branch of the UPR has been shown to be cyto-protective. In neurodegenerative diseases and other protein folding diseases, such as retinitis pigmentosa leading to blindness, cells die of UPR-induced apoptosis. The same concepts apply to many other diseases in which the UPR has been implicated.
The present invention provides unanticipated means to pharmacologically modulate (e.g., increase or decrease to varying degrees) the capacity of cells to fold proteins and prevent UPR-induced cell death. The first small molecule capable of modulating wild type Ire1 and its mode of action are presented. Furthermore, a robust and highly efficient assay to screen for target and mode of action of Ire1 modulators together with means to screen for new ones is provided.